Results

TGF-β1 induced a dose dependent inhibition of tip cell assignment and subsequent angiogenesis on Matrigel, maximal at 5.0 ng/ml. This occurred via ALK5-dependent pathways and was accompanied by significant upregulation of the TGF-β co-receptor endoglin, and SMAD2 phosphorylation, but no alteration in Smad1/5 activation. TGF-β1 also induced ALK5-dependent downregulation of Notch1 but not of its ligand delta-like ligand 4. Cell associated VEGFR2 (but not VEGFR1) was significantly downregulated and accompanied by reciprocal upregulation of VEGFR2 in conditioned medium. Quantitative polymerase chain reaction analysis revealed that this soluble VEGFR2 was not generated by a selective shift in mRNA isoform transcription. This VEGFR2 in conditioned medium was full-length protein and was associated with increased soluble HSP-90, consistent with a possible shedding of microvesicles/exosomes.

Conclusions

Taken together, our results suggest that endothelial cells exposed to TGF-β1 lose both tip and stalk cell identity, possibly mediated by loss of VEGFR2 signaling. The role of these events in physiological and pathological angiogenesis requires further investigation.

The cytokine transforming growth factor beta (TGF-β) is a member of the TGF-β superfamily consisting of 33 members including TGF-βs (1–3) [1]. TGF-β1 is the predominant and more ubiquitous form. TGF-β ligands signal canonically through type I (ALK) and type II serine/threonine kinase receptors [2], and via the accessory (type III) receptor endoglin in vascular endothelial cells [3]. TGF-β binding leads to phosphorylation of intracellular R-Smads 1, 2, 3, 5 or 8 [2, 4], which then complex with Co-Smad4, enter the nucleus and associate with transcriptional co-activators or co-repressors to regulate the expression of target genes.

TGF-β plays a dual role in angiogenesis by orchestrating a switch from vascular inhibition to pro-angiogenic activity [5–7]. In particular, there is evidence that TGF-β1 induced angiogenesis acts with VEGF to mediate apoptosis of excessive vascular sprouts and may even be required for initial sprouting from an existing vascular network [8, 9]. The nature of the angiogenic response to TGF-β depends on the balance of ALK1 versus ALK5 signaling input, with ALK1 predominantly promoting sprouting and ALK5 favoring the resolution/stabilization phase of angiogenesis [6]. Thus, TGF-β is either pro- or anti-angiogenic, depending on which TGF-RII/Smad pathway is engaged [10]. Differences also exist in the kinetics and dose responses of these pathways: ALK1 mediated signaling is transient and maximal at low concentrations of ligand while ALK5 mediated signaling is sustained and maximal at higher doses of ligand [11]. Increased expression of endoglin led to inhibition of TGF-β/ALK5 signaling (as demonstrated by Smad and CAGA dependent reporter activity) in a dose dependent fashion [12].

Activation of vascular sprouting during angiogenesis entails the specification of endothelial cells into tip and stalk cells. Endothelial tip cells are mainly migratory and polarized with minimal proliferation while stalk cells proliferate throughout sprout establishment and form the nascent vascular lumen cells [13]. The delta-like ligand 4 (Dll4)-Notch1 signaling pathway is involved in tip-stalk cell identity [14]. Tip cells express high levels of Dll4 and vascular endothelial growth factor receptor-2 (VEGFR2), and have low levels of Notch signaling activity [13, 15, 16]. The specification of endothelial cells as tip or stalk cells is transient and its reversibility is contingent on the balance between pro-angiogenic factors. Here we quantify the effect of TGF-β1 on in vitro angiogenesis using a Matrigel cord formation assay, and determine the impact of this angiogenic cytokine on expression of molecules associated with endothelial tip and stalk cell identity.

RNA isolation and gene expression analysis

Cells were lysed in Ribozol (AMRESCO, VWR International, Mississauga, ON) and RNA isolated using Aurum Total RNA columns (Bio-Rad, Mississauga, ON) according to manufacturers’ protocols. RNA was reverse-transcribed with 4 μL iScript (BioRad, Mississauga, ON) before amplification using primers for target and housekeeping genes (Table 1). Ssofast EvaGreen Supermix (BioRad) was used to determine primer efficiency and for quantitative PCR. No-template controls and quantitative PCR reactions were run in triplicate with an initial 2 min denaturation at 95 °C, 40 cycles of 95 °C for 5 s, 60 °C for 5 s, followed by melt curve analysis of 65 °C to 95 °C in 0.5 °C increments on the CFX96 RealTime System (Bio-Rad). Full length and soluble splice variant mRNA for VEGFR2 were first normalized to both HPRT and GAPDH housekeeping genes, and then results from TGF-β1-treated samples were expressed as relative amounts normalized to control (untreated) samples. Data were analyzed using CFX Manager (Bio-Rad).

Matrigel cord formation assay

The impact of TGF-β1 and inhibitors on in vitro angiogenesis was determined by cord formation assay. 10 μl of growth factor containing BD Matrigel Matrix (cat # 354234; BD Biosciences) was added to each well of cold angiogenesis μ-slides (ibidi) and allowed to gel at 37 °C for 30 min. 50 μl of BAEC cell suspension containing 1 × 104 cells in Media-200 made with complete supplements (GIBCO) plus or minus TGF-β1 and inhibitors were added to each well. Slides were incubated for 8 h at 37 °C, and one image/well was captured with phase contrast microscopy using a 4X objective. The generation of cord networks was quantified using the tube formation assay analysis service Wimtube (http://www.wimasis.com; ibidi); analyzed parameters were total cord length, # of branching points, and # of loops. # of tip cells was also determined by manual count of phase contrast images; tip cells were defined as free-ended cells/sprouts with visible filopodia.

Statistical analysis

All statistical analysis and graphing were performed using GraphPad Prism 6 software (GraphPad Software). Means for at least three biological replicates were calculated and plotted with standard error bars. Data were analyzed by using the nonparametric Kruskal-Wallis test followed by Dunn’s post hoc test for multiple comparisons, as appropriate. Differences between means were considered statistically significant when the p value was less than 0.05. Assays were performed in triplicate unless otherwise indicated.

The mechanisms of the observed changes in angiogenic signaling pathways were then investigated. As demonstrated by treatment with the ALK5 signaling inhibitor SB-431542 (SB, 5 μM), blockade of ALK5 signaling significantly eliminated the ability of 5.0 ng/ml TGF-β1 to inhibit Matrigel angiogenesis (Fig. 3 a). SB inhibitor alone had no significant effect compared to DMSO vehicle (Control) in these assays. Exogenous TGF-β1 led to a reduction in VEGFR2 phosphorylation (as revealed by western blotting for pVEGFR2 at residue tyrosine 951) and expression levels, but addition of the SB inhibitor rescued VEGFR2 signaling (Fig. 4 a). Similar results were obtained using SD-208, another ALK5 signaling inhibitor (Additional file 2: Figure S2). TGF-β1 activation of SMAD2 phosphorylation and endoglin upregulation was also blocked by SB (Fig. 4). Notch1 levels showed a trend towards significant differences with SB inhibitor (p = 0.07), but expression of Dll4 was not altered (Fig. 4). SMAD1/5 phosphorylation was also not altered in these samples. Thus, ALK5 appeared to mediate the effects of TGF-β1 on the observed altered angiogenesis signaling pathways.

The basis of VEGFR2 downregulation was further investigated. Loss of VEGFR2 from whole cell lysates of TGF-β1 treated BAEC was accompanied by concomitant increasing levels of full-length protein in serum free conditioned media (CM) collected from the same cells (Fig. 5 a). qPCR analysis showed that both full-length and the soluble VEGFR2 (sVEGFR2) splice variant mRNAs were expressed by these cells, with the full-length isoform being the predominant species, expressed approximately 16 fold higher than the sVEGFR2 message. Both isoforms of VEGFR2 message were significantly downregulated in TGF-β1 treated cells compared to control. However, the ratio of full-length to soluble VEGFR2 mRNA was not altered by 5.0 ng/ml TGF-β1 treatment (Fig. 5 b), supporting the finding from Fig. 5a that a truncated soluble form of VEGFR2 protein is not released from these cells upon TGF- β1 exposure.

No change in MMP-14 (MT1-MMP) or ADAM17 protein levels were detected, and ADAM10 was weakly upregulated in TGF-β1 treated cells, suggesting enzymatic shedding was not the mechanism for increased CM-associated VEGFR2 (Fig. 5 d). In contrast, increased levels of the extracellular vesicle/exosome-associated protein HSP90 were also detected in serum-free conditioned medium from TGF-β1 treated cells (Fig. 5 d). These data suggest that in addition to transcriptional downregulation, full-length VEGFR2 is released from TGF-β1 treated endothelial cells as a mechanism to regulate angiogenesis.

TGF-β-mediated ALK1 signaling serves to enhance sprouting angiogenesis in part by indirectly inhibiting TGF-β/ALK5 signaling [6]. Endoglin modulates the balance between ALK1 and ALK5 signaling, favouring a pro-angiogenic phenotype [23]. However, in our study, endoglin expression was highest in cells treated with ‘anti-angiogenic’ levels of TGF-β1 (5 ng/ml and higher), which were also associated with ALK5/Smad2 activation. Interestingly, activated (phosphorylated) Smad1/5 was readily detected independent of TGF-β1 treatment. Thus, in our system, tip cell identity may be the default phenotype, which is repressed upon activation of ALK5/Smad2 signaling, possibly via loss of Notch 1 and VEGFR2 expression.

We saw concomitant loss of cell-associated full-length VEGFR2 and increased levels of CM-associated full-length VEGFR2 upon TGF-β1 exposure. Endothelial cells are known to produce an alternatively spliced VEGFR2 mRNA, leading to transcripts lacking the transmembrane domain coded for by exon 13 [24, 25]. This transcript codes for a soluble form of the receptor (sVEGFR2), which could potentially account for the presence of CM-associated VEGFR2 in our samples. However, while both full length and alternatively spliced VEGFR2 transcripts were detectable in BAEC, the full length VEGFR2 mRNA was by far the predominant isoform, and we found no evidence for a preferential shift in production of message for sVEGFR2 in TGF-β1 treated cells. Further, the CM-associated VEGFR2 was full-length protein as assessed by estimated molecular mass. Thus, the source of VEGFR2 found in conditioned medium is unlikely to be due to preferential translation of a sVEGFR2 variant mRNA upon exposure to TGF-β1.

Alternatively, VEGFR2 could be cleaved from the BAEC cell surface through proteolytic activity. The matrix metalloproteinase MMP-14 (membrane type MMP; MT1-MMP) is known to cleave the TGF-β type III receptor endoglin from endothelial cells, generating a soluble form [26]. In our study, levels of MMP-14 were weakly detectable by western blotting, and qPCR revealed Cq values > 35 (data not shown). This, combined with the lack of detectable soluble endoglin in BAEC conditioned medium, suggests that MMP-14 cleavage of surface protein is not a significant determinant of CM-associated VEGFR2 in our system. A disintegrin and metalloprotease (ADAM) family of enzymes act as ‘sheddases’, cleaving surface bound proteins to general soluble isoforms. ADAM-17 is known to shed VEGFR2 from endothelial [27] and non-endothelial [28] cell surfaces, and VEGFR2 is a target of ADAM-10 mediated cleavage in endothelial cells [29]. Both ADAM-10 and ADAM-17 were produced by BAECs, and there was weak induction of ADAM-10 by TGF-β1 in our cells. However, such sheddase activity would generate CM-associated VEGFR2 molecules of ~130 kDa [27], but we only detected full length (~250 kDa) VEGFR2 in conditioned medium, consistent with an alternative mechanism for release of this receptor from endothelial cells. Recent reports suggest that prolonged surface residence of VEGFR2 in HUVECs leads to protease-mediated cleavage and the generation of a soluble fragment of ~100 kDa and a residual cell associated 130 kDa fragment [30]. As well, enhanced ubiquitination of VEGFR2 upon internalization in HUVECs leads to endosome-lysosome pathway mediated fragmentation into 160 and 120 kDa fragments [31, 32]. The absence of such VEGFR2 fragments in our samples suggests that TGF-β1 is not modulating VEGFR2 levels via these mechanisms in these BAECs.

In agreement with the aforementioned findings, our results extend this activity to include the possibility that shedding of VEGFR2 containing extracellular vesicles (possibly exosomes) by vascular endothelium may be enhanced upon exposure to TGF-β1. We detected full length VEGFR2 in serum free conditioned medium along with the exosome marker HSP90. Retinal pigment epithelial cells also shed exosomes containing VEGFR2 which subsequently modulate endothelial cord formation in vitro [45], but to our knowledge this is the first report that VEGFR2 might be shed from endothelial cells themselves.

Here we report that TGF-β1 alters levels of key angiogenic receptors, and interferes with tip/stalk cell identity when ALK5/Smad2 signaling pathways are activated. Our results suggest that downregulation of surface VEGFR2 and concomitant increase in VEGFR2 levels in endothelial cell conditioned medium may be a direct response to ALK5-mediated TGF-β signaling. Exosome shedding of VEGFR2 may rapidly limit the effects of angiogenic stimuli on the cells and stop further sprout formation, but the role of these events in physiological and pathological angiogenesis requires further investigation.

Acknowledgements

We thank other members of the Coomber and Viloria-Petit laboratories for their helpful suggestions.

Funding

This study was funded by the Nick Natale Innovation Grant of the Canadian Cancer Society (#2012-701069) to BLC and AVP. Infrastructure funds were provided by a Canadian Foundation for Innovation Grant #26472 to AVP. Personal support was provided to MJ by the Saudi Arabia Ministry of Higher Education. These funding bodies played no role in the design of the study, the collection, analysis, and interpretation of data or in writing the manuscript.

Availability of data and material

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Authors’ contributions

MJ, EAK and JM performed cell culture experiments, western blotting and data analysis; BLC performed cord formation assays and data analysis; all authors contributed to the writing of the manuscript and approved its final content.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable

Ethics approval and consent to participate

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